Enantioselective binding of disopyramide to α1-acid glycoprotein and its variants

OBJECTIVE: alpha1-acid glycoprotein (AAG) has three main genetic variants, F1, S, and A variants. There are few reports on the correlation between AAG variants and binding activity of drug enantiomers. We studied the differences between the binding characteristics of enantiomers of disopyramide (DP), which is a basic drug. The aim of this study was to elucidate the cause of the differences between the binding characteristics of DP enantiomers. METHODS: The variants in human AAG were separated by hydroxyapatite chromatography. Binding of DP enantiomers to AAG variants was studied by the ultrafiltration method. The characteristics of the binding of DP enantiomers to total variants and each variant were examined by Scatchard analysis within a range of concentrations from 0.5 to 50.0 microg/ml. RESULTS: The binding capacity of S-DP was significantly higher than that of R-DP in variant 3, although the binding capacities of DP enantiomers were almost the same in variant 2. On the other hand, the binding capacities for both S-DP and R-DP in variant 3 were significantly higher than those in variant 2. Furthermore, there was an almost 2.4-fold difference in the dissociation constant (Kd) between S-DP and R-DP in variant 3, although no significant difference was observed in the number of binding sites (N). In variant 2 no significant differences between DP enantiomers were observed in either the dissociation constant or number of binding sites per molecule of AAG. On the other hand, significant differences between variants 2 and 3 in the dissociation constant for both S-DP and R-DP were observed. The differences in dissociation constant between variants 2 and 3 were 4.0-fold in S-DP and 1.7-fold in R-DP. CONCLUSION: The difference between the binding capacities of S-DP and R-DP is due to differences in the association of DP to variants 3-6, and the role of the variants 1 and 2 in the binding of drugs to AAG is minor.     

Population pharmacokinetics,protein binding and antiarrhythmic effects of disopyramide enantiomers in arrhythmic patients

Disopyramide (DP) is widely used as an antiarrhythmic agent. The antiarrhythmic effects of its enantiomers differ from each other and its metabolism and protein binding are also stereoselective. Population pharmacokinetic parameters of DP racemate, enantiomers (S(+)-DP, R(-)-DP), and their unbound concentrations (uDP, S(+)-uDP and R(-)-uDP) were analyzed using the nonlinear mixed effect model (NONMEM) program. Data were available from 108 points of 33 arrhythmic patients on maintenance therapy with DP racemate. We evaluated the factors to which pharmacokinetic parameters are attributed and the relationships between each serum concentration and the antiarrhythmic effect. A one-compartment model was fitted to the data using NONMEM. For DP, S(+)-DP and R(-)-DP, elimination rate constants (kes) were estimated as 0.0648, 0.0663 and 0.0691/h, respectively and the mean apparent volume of distribution (Vd/F) were estimated as 63.2, 54.1 and 71.6 l, respectively. Using the ke and Vd/F values estimated by NONMEM, time-concentration curves were well fitted to the observed data. Unbound fractions of both DP enantiomers showed nonlinearity and the binding ratio of S(+)-DP was 0.84 +/- 0.07, which was higher than that of R(-)-DP [0.70 +/- 0.11 (p < 0.01)]. Unbound fractions of both DP enantiomers correlated with alpha1-acid glycoprotein (AGP) (p < 0.01). On the other hand, using NONMEM, a significant proportion of the variability of Vd/F could be attributed only to AGP (p < 0.001). NONMEM was able to clarify the pharmacokinetic features in the protein binding of DP. Individual steady state concentrations were estimated by NONMEM using the Bayesian method. The average unbound concentrations of all nine responders were higher than those of the four non-responders, even though this difference was not significant. Unbound concentrations may reflect drug concentrations in the tissue, which suggests that these concentrations may indicate an antiarrhythmic effect rather than the total concentration.     

Stereoselective metabolism and pharmacokinetics of disopyramide enantiomers in humans

Metabolism, pharmacokinetics, and influence of alpha 1-acid glycoprotein (alpha 1-AGP) plasma levels on protein binding of (R)-(-) and (S)-(+)-disopyramide (DP) were compared, in six healthy subjects, at the steady state, after oral administration of 100 mg twice daily. The mean unbound clearance of (R)-(-)-DP and (S)-(+)-DP were 8.59 and 14.9 ml/min/kg, respectively (p = 0.003). The mean unbound renal clearance of (R)-(-)-DP and (S)-(+)-DP were 6.26 and 8.75 ml/min/kg, respectively (p = 0.025). The nonrenal clearance, i.e. hepatic metabolic clearance, of (R)-(-)-DP and (S)-(+)-DP averaged 2.32 and 6.19 ml/min/kg, respectively (p = 0.002). The mean unbound volume of distribution of (R)-(-)- and (S)-(+)-DP were 225 and 381 liters, respectively (p = 0.023). The half-life of (R)-(-)-DP and (S)-(+)-DP averaged 4.17 and 3.91 hr, respectively (p = 0.21). The mean unbound renal clearance of (R)-(-)- and (S)-(+)-mono-N-dealkylated disopyramide (MND) were 3.21 and 7.02 ml/min/kg, respectively (p less than 0.001). The unbound fraction at steady state of (R)-(-)-DP and (S)-(+)-DP averaged 12.5 and 7.5%, respectively (p = 0.002). The unbound fraction at steady state of (R)-(-)-DP and (S)-(+)-MND averaged 62.6 and 60.5%, respectively (p = 0.36). The highest alpha 1-AGP plasma concentration resulted in lower unbound fraction for both DP and MND enantiomers, whereas the lowest alpha 1-AGP plasma concentration resulted in higher unbound fraction for (S)-(+)-DP only.     

Age- and gender-related differences in carbohydrate concentrations of alpha1-acid glycoprotein variants and the effects of glycoforms on their drug-binding capacities

OBJECTIVE: Alpha(1)-acid glycoprotein (AAG) is a major binding protein for neutral and basic drugs because of its great drug affinity. AAG has three main genetic variants--F1, S, and A variants. Several attempts have been made to elucidate the differences in compositions of the carbohydrate moiety and structure-function relationships such as drug-binding differences. However, there have been few reports on age- and gender-related differences in compositions or concentrations of the carbohydrate moiety of AAG variants. The aim of this study was to clarify the age- and gender-related differences in carbohydrate concentrations and in drug-binding capacities of AAG glycoforms. METHODS: The sera used in this study were obtained from 32 healthy subjects (17 men and 15 women, aged 16-84 years). The AAG glycoforms were isolated by hydroxyapatite chromatography. The binding capacity of AAG to disopyramide (DP), which is a basic drug, was determined using the ultrafiltration method. The concentrations of N-acetylneuraminic acid (NeuAc) and monosaccharides in AAG were determined using high-pH anion-exchange chromatography with pulsed-amperometric detection. RESULTS: The mean plasma AAG concentration in the female subjects was significantly lower than that in the male subjects (0.67 +/- 0.12 mg/ml, mean +/- SD, in females, n = 15, versus 0.81 +/- 0.17 mg/ml in males, n = 17, P < 0.05), but no age-related differences were found (0.75 +/- 0.18 mg/ml in young subjects, n = 24, versus 0.77 +/- 0.12 mg/ml in older subjects, n = 8, n.s.). However, the degree of branching of the glycan chain in the female subjects was significantly lower than that in the male subjects (1.61 +/- 0.17 mol/mol, mean +/- SD, in females, n = 15, versus 1.75 +/- 0.23 mol/mol in males, n = 17, P < 0.05). There was a significant inverse relationship between the binding capacity of AAG to DP (Cb/AAG) and the degree of branching of the glycan chain. The binding capacity (Cb/AAG) decreased as the degree of branching in AAG glycans increased. The binding capacity (Cb/AAG) in the female subjects was significantly higher than that in the male subjects (2.79 +/- 0.59 mg/g AAG in females, mean +/- SD, n = 15, versus 2.37 +/- 0.29 mg/g AAG in males, n = 17, P < 0.05). CONCLUSION. The degree of branching of the glycan chain in AAG plays an important role in drug-binding capacity. Gender-related differences in drug-binding capacity (Cb/AAG) may be caused by differences in the ratios of the extent of branching of the glycan chain in AAG.     

Stereoselectivity in the metabolism of disopyramide enantiomers in rat and dog

The metabolism and pharmacokinetics of (S)-(+)- and (R)-(-)-disopyramide (DP) were compared in laboratory animals. In rats, after oral administration of (S)-(+)-DP phosphate salt at a dose of 38.8 mg of DP free base per kg, the mean maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) were 1.43 micrograms/ml and 4.47 micrograms . hr/ml, respectively, and after (R)-(-)-DP administration, the values were 4.93 micrograms/ml and 17.05 micrograms . hr/ml, respectively. Similar recoveries (approximately 55% of the dose) of DP and its metabolites in the urine and bile of rats were obtained after administration of the individual (S)-(+)- and (R)-(-)-enantiomers of DP. These results indicate similar oral absorption of the two enantiomers, but greater metabolism with (S)-(+)-DP in rats. In dogs, the AUC of DP after iv administration of (R)-(-)-DP phosphate salt at a dose of 15 mg of DP free base per kg was 1.6 times greater than that after (S)-(+)-DP phosphate salt. Inasmuch as volumes of distribution of the two enantiomers were similar, this difference can be attributed to a difference in the elimination rates of the enantiomers. After an oral dose, the difference in AUC of the two enantiomers was greater than that after an iv dose. The mean values of Cmax and AUC after a 15-mg/kg oral dose of (S)-(+)-DP were 1.32 micrograms/ml and 4.07 micrograms . hr/ml, respectively, and with (R)-(-)-DP the values were 2.88 micrograms/ml and 9.21 micrograms . hr/ml, respectively. The Cmax and AUC of the total drug-related materials (drug plus its metabolites) were similar. These results, together with the similarity in the urinary excretion of total drug related compounds, indicated that after equivalent doses of the two enantiomers, oral absorptions were similar, but the first-pass metabolism was greater with (S)-(+)-DP. The present study also demonstrated that N-dealkylation in dogs and arylhydroxylation in rats are stereoselective metabolic pathways, thus illustrating species differences in the stereoselective metabolism of DP.     

Protein binding of propranolol and verapamil enantiomers in maternal and foetal serum.

The protein binding of the enantiomers of propranolol and verapamil was measured in 19 pairs of maternal and foetal serum obtained at delivery. The binding of the enantiomers of both drugs was lower in foetal than in maternal serum. In maternal serum the mean (+/- s.d.) unbound percentages were 22.4 +/- 6.2 and 20.7 +/- 6.6 for R- and S-propranolol, and 16.8 +/- 5.5 and 22.5 +/- 6.2 for R- and S-verapamil; in foetal serum the values were 38.8 +/- 8.6 and 40.4 +/- 10.6 for R- and S-propranolol, and 34.7 +/- 10.5 and 44.8 +/- 10.7 for R- and S-verapamil. For propranolol, in maternal, but not in foetal serum, the difference between the binding of the R- and S-enantiomers was significant; the R/S ratio was significantly (P < 0.01) larger in the mother (1.099 +/- 0.072) than in the foetus (0.973 +/- 0.068). For verapamil, the difference in binding between the R- and S-enantiomers was significant in both maternal and foetal serum, but the R/S ratio was similar in mother (0.735 +/- 0.098) and foetus (0.763 +/- 0.070). Serum alpha 1-acid glycoprotein (AAG) concentrations were markedly higher and albumin concentrations slightly lower in maternal than in foetal samples. The binding of the four enantiomers in maternal and foetal serum was correlated (P < 0.001) with the AAG concentration (r propranolol: R 0.749, S 0.746; r verapamil: R 0.753, S 0.782). Our findings show that measurement of concentrations of total, unresolved drug allow a reasonably accurate assessment of transplacental gradients of individual isomers.     

Identification of CYP3A4 as the enzyme involved in the mono-N-dealkylation of disopyramide enantiomers in humans.

To identify which cytochrome P-450 (CYP) isoform(s) are involved in the major pathway of disopyramide (DP) enantiomers metabolism in humans, the in vitro formation of mono-N-desalkyldisopyramide from each DP enantiomer was studied with human liver microsomes and nine recombinant human CYPs. Substrate inhibition showed that SKF 525A and troleandomycin potently suppressed the metabolism of both DP enantiomers with IC50 values for R(-)- and S(+)-DP of <7.3 and <18.9 microM, respectively. In contrast, only weak inhibitory effects (i.e., IC50 > 100 microM) were observed for five other representative CYP isoform substrates [i.e., phenacetin (CYP1A1/2), sparteine (CYP2D6), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), and p-nitrophenol (CYP2E1)]. Significant correlations (P <.01, r = 0.91) were found between the activities of 11 different human liver microsomes for mono-N-dealkylation of both DP enantiomers and that of 6beta-hydroxylation of testosterone. Conversely, no significant correlations were observed between the catalytic activities for DP enantiomers and those for the O-deethylation of phenacetin, 2-hydroxylation of desipramine, hydroxylation of tolbutamide, and 4'-hydroxylation of S-mephenytoin. Further evidence for involvement of CYP3A P450s was revealed by an anti-human CYP3A serum that inhibited the mono-N-dealkylation of both DP enantiomers and 6beta-hydroxylation of testosterone almost completely (i.e., >90%), whereas it only weakly inhibited (i.e., <15%) CYP1A1/2- or 2C19-mediated reactions. Finally, the recombinant human CYP3A3 and 3A4 showed much greater catalytic activities than seven other isoforms examined (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1, and 3A5) for both DP enantiomers. In conclusion, the metabolism of both DP enantiomers in humans would primarily be catalyzed by CYP3A4, implying that DP may have an interaction potential with other CYP3A substrates and/or inhibitors.     

Enantioselective binding of propranolol, disopyramide, and verapamil to human alpha(1)-acid glycoprotein

We investigated the binding of propranolol (PL), disopyramide (DP), and verapamil (VP) enantiomers by human alpha(1)-acid glycoprotein (AGP; also called orosomucoid) and the relationships between the extent of drug binding and lipophilicity, desialylation, and genetic variants of AGP. Desialylation had little effect on the affinity of AGP for the drugs tested. The percentage binding correlated significantly with the partition coefficients for the drugs tested. Each enantiomer was competitively displaced from AGP by another enantiomer of the same drug, suggesting that they bind to the same site. However, the enantiomers bound to AGP with stereospecific affinities; the (-)-isomers of DP and VP had higher Kd values (4.27 and 4.97 microM, respectively) than the (+)-isomers (1.51 and 2.48 microM, respectively). When enantiomers of the different drugs were used in competitive binding experiments, VP binding was only partially inhibited by DP. This result suggested that drug binding is specific to different variants of AGP (A, F1, S). DP was found to specifically bind to variant A, whereas PL and VP bind to both A and F1/S variants.     

Plasma protein binding of penbutolol in pregnancy

Penbutolol is a not cardioselective beta-adrenergic blocking drug; it is lipid soluble and differs in its protein binding from the other members of its group because shows linkage to alpha 1-glycoprotein, with no detectable binding to albumin. AAG levels change during pregnancy and so the binding of [3H]-penbutolol was compared in 11 pregnant patients and in 10 healthy women. Binding was obtained by ultrafiltration and measurement of the free fraction by scintillation spectrometry. The free penbutolol fraction was significantly higher in the pregnant women than in the controls (6.06 +/- 0.34 compared with 3.55 +/- 0.29, P less than 0.001). The AAG levels in the pregnant women were significantly lower (0.40 +/- 0.03 g/l) than in the controls (0.77 +/- 0.06 g/l) (P less than 0.001) which showed a significant correlation with the bound/free penbutolol ratio (r = 0.61, P less than 0.005). On the other hand there was no significant correlation with the extent of penbutolol's protein binding even though the albumin levels were lower in the pregnant women (2.83 +/- 0.17 compared with 4.86 +/- 0.17; P less than 0.001). Penbutolol's nK1a for AAG was lower in pregnant women, and this suggests that the fall in AAG levels is not the only factor involved in the reduced binding of penbutolol in pregnancy.     

Binding of catecholamines to alpha-1 acid glycoprotein, albumin and lipoproteins in human serum

The binding of catecholamines in human serum was determined by equilibrium dialysis at 37 degrees. For serum concentrations of 10-15 nM the bound fractions were 28.8 +/- 2.2%, 25.7 +/- 1.7% and 22.2 +/- 2.2% for (+/-)-isoproterenol (IPR), (+/-)-norepinephrine (NE) and (+/-)-epinephrine (EPI), respectively. At higher serum concentrations saturation occurred. Alpha-1 acid glycoprotein (AAG) possessed one high affinity binding site and approximately 10 low affinity sites. The catecholamines were bound to AAG with the same order of potency for both classes of binding sites: IPR (Kd1: 100 microM Kd2: 2.2 mM) greater than NE (Kd1: 120 microM, Kd2: 6.5 mM) greater than EPI (Kd1: 140 microM, Kd2: 14 mM). Human serum albumin (HSA) and lipoproteins (SLP) interacted with the catecholamines in a non-saturable manner. IPR showed the strongest and EPI the weakest association to both of these serum protein fractions. (-)-Propranolol was able to inhibit the binding of IPR in serum and to isolated AAG, but not to HSA or to SLP. The present results show that AAG is an important catecholamine-binding protein in human serum. AAG, but not HSA or SLP, possesses binding sites shared by adrenergic receptor stimulators and blockers.     

Relationship between alpha 1-acid glycoprotein and plasma binding of disopyramide and mono-N-dealkyldisopyramide.

Highly purified serum albumin did not bind either disopyramide (DP) or mono-N-dealkyldisopyramide (MND). The unbound fraction of DP and MND in highly purified serum alpha 1-acid glycoprotein (AAG) at 0.5 g/l was 57 and 62 and at 2.0 g/l 19 and 30% respectively. Unbound DP and MND were measured in spiked plasma (10 mumol/l of DP or MND), from 60 patients, having AAG concentrations varying from 0.4 to 3.0 g/l. Unbound drug varied from 13 to 58 and from 24 to 62% for DP and MND, respectively, and was inversely related to the plasma concentration of AAG (r = -0.9016, r = -0.9157). A linear relationship was found between the binding ratio (moles bound divided by moles unbound) and the plasma concentration of AAG for both DP (r = 0.9199) and MND (r = 0.9270), whereas no relationship was found between the binding ratios of DP or MND and the plasma concentrations of total protein, albumin, haptoglobin, alpha 1-antitrypsin or the immunoglobulins IgG, IgA or IgM. In patients on DP maintenance therapy, a linear relationship was found between percent unbound DP and the plasma concentration of DP in samples with similar AAG concentrations. Furthermore, a linear relationship was found between the binding ratio of DP and the plasma concentration of AAG in samples with similar DP concentrations. The present findings support the concept that AAG is the major serum protein responsible for the binding of DP and MND.     

Protein binding of nifedipine

The protein binding of nifedipine in concentrations up to 1200 ng ml-1 has been measured in serum, pure human albumin solution and pure human alpha 1-acid glycoprotein (AAG) solutions by ultrafiltration. The drug was extensively bound in serum from four healthy volunteers with a mean (+/- s.d.) fraction bound of 0.992 +/- 0.008. In albumin solution (40 g litre-1) the mean (+/- s.d.) fraction bound 0.970 +/- 0.012, was not significantly different (P greater than 0.05) from that in serum, suggesting that albumin is the major, but not necessarily the only, binding protein for nifedipine in serum. The binding of nifedipine in solutions of AAG was proportional to the AAG concentration and ranged from 0.514 +/- 0.059 to 0.755 +/- 0.035 in solutions containing 50 and 150 mg % AAG, respectively. Binding of nifedipine in all protein solutions was linear.     

Stereoselective binding of propranolol in the elderly.

The influence of age on the stereoselective serum protein binding of propranolol was investigated. Serum was obtained from 10 young (mean age 23 +/- 2 years) and ten elderly (mean age 68 +/- 2 years) healthy male volunteers. The free fraction of propranolol (fu) was measured by equilibrium dialysis, using tritium labeled (+/-)- and (-)-propranolol. The fu values were 0.159 +/- 0.049 and 0.148 +/- 0.028 (+/-), 0.135 +/- 0.041 and 0.136 +/- 0.04 (-), 0.174 +/- 0.056 and 0.161 +/- 0.028 (+) in the young and elderly subjects, respectively. Serum alpha 1-acid glycoprotein (AAG) concentrations were 81.4 +/- 33.0 and 81.0 +/- 21.6 mg 100 ml-1 in young and elderly respectively (P greater than 0.05). Variability in AAG concentration accounted for most of the observed intersubject variability in the binding of both propranolol isomers. The stereoselective binding of propranolol does not appear to be affected by age.     

Fluorescence studies of beta-adrenergic ligand binding to alpha 1-acid glycoprotein with 1-anilino-8-naphthalene sulfonate, isoprenaline, adrenaline and propranolol.

The present study shows that ANS (1-anilino-8-naphthalene sulfonate), propranolol, isoprenaline, adrenaline and dopamine have common binding sites on AAG (alpha 1-acid glycoprotein). A fluorescence technique was employed to characterize the interaction between the ligands and AAG at 20-22 degrees. The binding of ANS to AAG caused increased fluorescence intensity at emission and excitation wavelengths of 400 and 470 nm. In this situation, propranolol displaced ANS in a concentration-dependent mode with an apparent dissociation constant of 6.2 +/- 0.01 microM, whereas isoprenaline did not reduce the ANS-AAG fluorescence. However, in the presence of AAG, catecholamines caused a marked increase of fluorescence at excitation and emission wavelengths of 250 and 325 nm, respectively. These wavelengths were employed to characterize the binding of isoprenaline, adrenaline and propranolol to AAG. Two subsets of binding sites were demonstrated. The Kd values were 0.87 +/- 0.03 and 25.1 +/- 10.7 microM for ANS, 0.76 +/- 0.09 and 133 +/- 30.4 microM for propranolol, 140 +/- 14 and 2.18 +/- 0.58 mM for isoprenaline, 137 +/- 24 and 14.8 +/- 0.1 mM for adrenaline, respectively. AAG had identical high affinity binding capacity for these ligands (n approximately 1). However, the second class of binding sites showed ligand-dependent binding capacity: n = 1 for ANS, n approximately 10 for propranolol, n approximately 15 for adrenaline, n approximately 20 for isoprenaline, respectively. ANS, propranolol, dopamine and adrenaline caused concentration-dependent inhibition of isoprenaline binding to AAG with apparent dissociation constants of 5.1 +/- 1.8 microM, 6.4 +/- 1.1 microM, 0.57 +/- 0.13 mM and 1.5 +/- 0.46 mM, respectively.     

Interindividual differences in the binding of antidepressives to plasma proteins: the role of the variants of alpha1-acid glycoprotein

Summary Alpha₁-acid glycoprotein (AAG) is one of the plasma proteins that bind basic drugs, like amitriptyline (AT) and its metabolite nortriptyline (NT). Two types of genetic polymorphism have been described for AAG: polymorphic forms which, on electrophoresis of the native protein, give four patterns with 5, 6, 7 or 8 bands, and the variants which on by electrophoresis of the desialysed protein, give three patterns with 2 bands, FF, FS and SS. In 31 depressive patients, treated daily with 150 mg AT for 3 weeks, free and total plasma AT and NT were determined, as well as the AAG polymorphic forms and variants. There was only a weak negative correlation between the free fractions of AT and NT and total plasma AAG, but free AT and NT were strongly correlated with the S form (but not the F form) of AAG variants. The differences in binding might be the expression of a further genetic factor determining the steady-state plasma levels of tricyclic drugs.     

Pharmacology and autoradiography of human DP prostanoid receptors using [(3)H]-BWA868C, a DP receptor-selective antagonist radioligand

1. A potent and highly selective DP prostanoid receptor antagonist radioligand, [(3)H]-cyclohexyl-N-BWA868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethyl-amino) hydantoin, ([(3)H]-BWA868C)), has been generated for receptor binding and autoradiographic studies. 2. Specific [(3)H]-BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23 degrees C and constituted up to 95% of the total binding. The association (K(+1)) and dissociation (K(-1)) rate constants of binding were 0.758+/-0.064 min(-1), mmol and 0.0042+/-0.0002 min(-1), respectively, yielding dissociation constants (K(D)s) of 5.66+/-0. 44 nM (n=4). 3. Specific [(3)H]-BWA868C bound to DP receptors with a high affinity (K(D)=1.45+/-0.01 nM, n=3) and to a finite, saturable number of binding sites (B(max)=21.1+/-0.6 nmol g(-1) wet weight). 4. DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD(2)) exhibited high (nanomolar) affinities for [(3)H]-BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. 5. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [(3)H]-BWA868C. While [(3)H]-PGD(2) yielded similar quantitative distribution of DP receptors as [(3)H]-BWA868C, the level of non-specific binding observed with [(3)H]-PGD(2) was significantly greater than that observed with [(3)H]-BWA868C. 6. It is concluded that [(3)H]-BWA868C is a high-affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [(3)H]-BWA868C may prove useful for future homogenate-based and autoradiographic studies on the DP receptor.     

The pharmacokinetics of the enantiomers of mexiletine in humans

1. This study examined the pharmacokinetics of the enantiomers of mexiletine in five healthy subjects who were each given a single, 300 mg, oral dose of racemic mexiletine hydrochloride. 2. The time course of the concentration ratio between the R(-) and the S(+) enantiomers (R/S) in plasma showed a progressive decrease, with a mean +/- S.D. ratio of 1.37 +/- 0.11 at 1 h and 0.64 +/- 0.11 at 48 h. Similarly, the R/S ratios in urine were 1.38 +/- 0.42 and 0.55 +/- 0.12 at 1 h and 72 h, respectively. 3. The terminal elimination half-life of S(+)mexiletine was 11.0 +/- 3.80 h, which was significantly greater (P less than 0.05) than that of the R(-) enantiomer, 9.10 +/- 2.90 h. S(+)Mexiletine also showed a significantly greater apparent volume of distribution (P less than 0.01) and renal clearance (P less than 0.05) than R(-)mexiletine. There was no significant difference in the apparent oral total drug clearance of the enantiomers. 4. The disposition of mexiletine enantiomers in man was stereoselective, and the differences observed between the enantiomers may be due largely to differences in their serum protein binding.     

Use of pseudoracemic nitrendipine to elucidate the metabolic steps responsible for stereoselective disposition of nitrendipine enantiomers.

1. The pharmacokinetics, protein binding, bioavailability and metabolism of (+)-R- and (-)-S-nitrendipine were studied in six healthy subjects following random oral administration of 20 mg (+)-R-, 20 mg (-)-S- and 20 mg R,S-nitrendipine (pseudoracemic mixture of 10 mg [13C4)-(+)-R- and 10 mg (-)-S-enantiomer). 2. After administration of the enantiomers pronounced differences in AUC (R: 29.9 +/- 20.1; S: 123.8 +/- 63.7 ng ml-1 h; P less than 0.05), bioavailability (R: 10.7 +/- 7.4%; S: 44.6 +/- 23.1%; P less than 0.05) and Cmax (R: 14.4 +/- 7.7; S: 72.5 +/- 40.5 ng ml-1; P less than 0.05) were observed between R- and S-nitrendipine. When racemic nitrendipine was given bioavailability and dose normalized AUC and Cmax values of the S-enantiomer were not different from the values after S-nitrendipine-administration. In contrast, bioavailability (R: 10.7% R,S: 22.1%) and dose normalized AUC (R: 15.0; R,S: 29.5 ng ml-1 h and Cmax (R: 7.2; R,S: 16.8 ng ml-1) of R-nitrendipine were doubled following R,S- as compared with R-nitrendipine administration. t1/2 (R: 9.8; S: 9.1 h) and tmax were not different between the enantiomers nor were the values different after administration of the enantiomers or racemate. The fraction unbound in serum of R-nitrendipine was 0.0098 +/- 0.0032 (s.d.) and that of S-nitrendipine was 0.0083 +/- 0.0015 (s.d.). 3. The AUC values of the major pyridine metabolite M1 were similar after administration of R- and S-nitrendipine (S: 114.7 +/- 48.5; R: 71.7 +/- 29.9 ng ml-1 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of alpha-1-acid glycoprotein administration on propranolol binding and beta blockade in rats

Alpha-1-acid glycoprotein (AAG), 750 mg/kg, was administered to rats to determine its effect on propranolol binding and beta blockade. Anesthetized rats received [3H]propranolol i.v., followed in 15 min by human AAG or bovine serum albumin, 750 mg/kg. AAG treatment produced a human AAG concentration in serum of 7.76 +/- 1.17 mg/ml, several times higher than the endogenous serum AAG concentration in stressed rats. AAG treatment significantly increased the heart rate response to isoproterenol, compared to albumin (95.4 +/- 19.6 vs 28.3 +/- 16.7% of baseline value, measured 45 min after propranolol, P less than 0.001). AAG-treated rats had greater [3H]propranolol binding in serum (93.0 +/- 3.2 vs 76.7 +/- 3.0%, P less than 0.01) and a lower calculated unbound [3H]propranolol concentration in serum (2.7 +/- 1.3 vs 7.4 +/- 3.1 X 10(6) dpm/ml, P less than 0.001) than albumin-treated rats. These data demonstrate that AAG can alter propranolol pharmacokinetics and pharmacodynamics even when administered after the propranolol effect is evident. Because the reported affinity of propranolol for cardiac beta receptors is 10,000 times greater than its affinity for AAG, these data suggest that AAG acted by altering propranolol disposition rather than by directly competing with beta receptors for drug.     

Evidence for differences in the binding of drugs to the two main genetic variants of human alpha 1-acid glycoprotein.

1. Human alpha 1-acid glycoprotein (AAG), a plasma transport protein, has three main genetic variants. F1. S and A. Native commercial AAG (a mixture of almost equal proportions of these three variants) has been separated by chromatography into variants which correspond to the proteins of the two genes which code for AAG in humans: the A variant and a mixture of the F1 and S variants (60% F1 and 40% S). Their binding properties towards imipramine, warfarin and mifepristone were studied by equilibrium dialysis. 2. The F1S variant mixture strongly bound warfarin and mifepristone with an affinity of 1.89 and 2.06 x 10(6) l mol-1, respectively, but had a low affinity for imipramine. Conversely, the A variant strongly bound imipramine with an affinity of 0.98 x 10(6) l mol-1. The low degree of binding of warfarin and mifepristone to the A variant sample was explained by the presence of protein contaminants in this sample. These results indicate specific drug transport roles for each variant, with respect to its separate genetic origin. 3. Control binding experiments performed with (unfractionated) commercial AAG and with AAG isolated from individuals with either the F1/A or S/A phenotypes, agreed with these findings. The results for the binding of warfarin and mifepristone by the AAG samples were similar to those obtained with the F1S mixture: the mean high-affinity association constant of the AAG samples for each drug was of the same order as that of the F1S mixture: the decrease in the number of binding sites of the AAG samples, as compared with the F1S mixture, was explained by the smaller proportion of variants F1 and/or S in these samples. Conversely, results of the imipramine binding study with the AAG samples concurred with those for the binding of this basic drug by the A variant, with respect to the proportion of the A variant in these samples.